The phenotypic characterization of naturally occurring murine cytotoxic cells has been further developed to understand the origin, differentiation, and normal function of this population. Cells from spleen, lthymus, blood, and bone marrow have been characterized. Effector cell activity has been monitored against appropriate targets for both natural (NK) activity and natural cytotoxic (NC) activity. Their phenotype has been monitored by immunofluoresence (IF) with flow cytometry and by complement (C)-mediated, antibody dependent cytotoxicity. Splenic subpopulations enriched for large granular lymphocytes (LGL) from nylon wool nonadherent cells subjected to density separation techniques have been characterized with a series of monoclonal antibodies to T-cell antigens, to myelocytic antigens, and to other hematopoietic subsets. A subpopulation of low density splenic lymphocytes which is enriched both for cells with the morphology of LGL and NK function also contains a unique population of cells identified by two-parameter analysis for Ly 5 antigen expression and cell size. This population appears to be distinct from other nylon wool nonadherent lymphocytes in the spleen and will be further characterized for NK and NC activity. Thymic lymphocytes with a phenotype similar to that of some NK cells (i.e., low Ly 1, low Thy-1 antigen expression) are being further characterized for functional activity and for the ability to develop into NK cells.